Since this grant was first funded in 1985, it has been our goal to understand the molecular and cellular pathology of squamous cell cancers (SCC) of the aerodigestive tract, with the intent to develop novel molecular diagnostic, prognostic as well as therapeutic tools to improve the outcome of patients with these diseases. Since our discovery that most SCC cell lines are refractory to Transforming Growth Factor-I_ (TGFl_)-mediated cell cycle arrest, we have used a combination of molecular and immunohistochemical approaches to show that loss of expression and/or mutational inactivation of TGFI_ receptors, or the downstream mediators Smad2 and Smad4, results in global loss of TGFI_ responsiveness in a limited subset of SCCs. These studies firmly established TGF_'s role as a tumor suppressor. However, for most cases, the mechanisms of escape from TGFl_-mediated growth control remain unclear. Our first objective is to identify the underlying molecular mechanisms that account for loss of Smad2 activation or Smad4 deficiency found in a subset of human SCCs. Based on the key role Smad4 plays in mediating TGF_'s tumor suppressive function, our second objective is to identify Smad4's transcriptional targets and which of these mediate Smad4's tumor suppressive function. The second part of this grant tests the hypothesis that, during cancer development, TGFI_'s growth arrest function can become uncoupled from its ability to induce epithelial-to-mesenchymal transdifferentiation (EMT). In fact, under these conditions, constitutive activation of TGF_ signaling may enhance invasion and metastasis. However, little is known about the mechanisms that determine TGFI_ response specificity. Our objective is to test the hypothesis that TGFI_ signal strength, the kinetics of activation, nucleo-cytoplasmic shuttling and dephosphorylation of the two R-Smads, Smad2 and -3, and their differential associations with Smad4 and other cellular proteins are major determinants of TGFI_ cellular response specificity, and that their disruption results in uncoupling of the growth arrest function from EMT seen in cancer. We are uniquely positioned to address these questions because of the phospho-Smad2 and -3 antibodies that we have produced, our access to highly selective and potent inhibitors of the T_R-I kinase, and large scale tissue arrays of human SCC specimens to rapidly validate key experimental findings.